This R03 application is associated with the K08 grant entitled "Mechanisms of AIIograft Tolerance" that involves studying transplantation tolerance in a model utilizing murine islet allografts. In the first two years of in the original K08 grant period, several Specific Aims have either been completed or are well underway. The original focus of the study was to determine whether different therapies known to be effective in islet allograft prolongation worked by operationally similar or distinct immunological pathways. In the course of completing the 1st Specific Aim which examined the ability of tolerant spleen cells to coadoptively transfer tolerance (i.e., transfer tolerance despite the presence of equal numbers of naive spleen cells) to immunodeficient mice, it became clear that one therapy, in particular, developed so-called 'dominant' tolerance. Combined therapy monoclonal antibody therapy with anti-CD154 and anti-LFA-1 therapy not only led to exceptional islet allograft survival rates in 'high-responder' C57BL/6 recipients but also led to dominant transplantation tolerance in contrast to monotherapy with either anti-CD154, anti-CD154 with donor specific transfusion or anti-LFA-1. Therefore the original hypothesis that different therapies would lead to a similar form of tolerance was not bourn out with these studies. In fact, anti-CD154 and anti-LFA-1 appear to uniquely synergize and lead to a powerful form of regulatory tolerance. Given that both anti-CD154 and anti-LFA-1 are available in humanized forms, are non-lymphocyte depleting, and result in sustainable tolerance, the clinical applicability of this therapy for islet transplantation appears to be high. Because of the significance of these results, the remainder of the K08 period will include an evaluation of the dominant transplantation tolerance which results from combined anti-CD154/anti-LFA-1 therapy. The project has adapted the original studies in two other significant ways: (1) the role of NK and NK T cells will be evaluated with respect to the maintenance of dominant tolerance and (2) a new collaboration with Cytomation, Inc. will allow a more thorough assessment of graft infiltrating cells utilizing state-of-the-art flow cytometry and cell sorting techniques. The funds made available through this grant would assist the completion of Specific Aims mainly by providing for a full-time technician and by assisting with the significant animal costs associated with these studies.